The topic of the present study is the establishment of plasmid-mediated, mutation assay systems which allow us to determine the primary and higher level structural changes in DNA associated with mutation. These changes are generated by chemical and physical effects on DNA in vitro and in prokaryotic and eukaryotic cells. Pretreatment of plasmid DNA with carcinogens prior to transfection into E. coli. or eukaryotic cells permits us to assay mutation with no toxic effect on the host cells. The plasmid used for the study consisted of a combination of at least two well-defined genes, one of which is used for transformation selection and the other for mutation assay. Mutated plasmid DNA, isolated and cloned individually in large quantities, is investigated for determination of the chemical nature of DNA modification (such as sequence alteration). The altered genetic functions of mutated plasmids were tested by repeated transfection of the DNA and by subcloning of specific segments of DNA in other normal vector DNA.